Field of the Invention
The present invention pertains to novel nucleic acid fragments coding for insecticidal proteins. More specifically, the invention relates to novel fragments encoding insecticidal proteins, said proteins having greater solubility characteristics, less haemolytic activity, and/or greater expression potential in certain specific cells, than the protein encoded by the wild type 27 kDa Bacillus thuringiensis var. israelensis gene, the encoded proteins, insecticidal compositions containing these proteins, and th use of these proteins in combating insects, particularly mosquitoes, are also contemplated in the subject invention. Chimeric genes containing the novel nucleic acid fragments, and microorganisms, plant cells, plant tissues, seeds and plants incorporating the nucleic acid fragments are further within the ambit of the present invention.
The spore-forming bacteria Bacillus thuringiensis var. israelensis produces a proteinaceous crystalline inclusion which is toxic to the larvae of Mosquito News, 37: 355-358 (1977); de Barjac et al., CR Acad. Sci. Paris, ser D 286: 797-800 (1978); Thomas, et al., FEBS Letters, 154: 362-368 (1983). The native var. israelensis crystal is irregular in shape and consists of several major polypeptides in addition to a number of other polypeptides which are present in minor amounts. See, Thomas et al., J. of Cell Sci., 60: 181-197 (1983). A protein of molecular weight 27 kDa is the most prominent of these polypeptides, and its larvicidal and haemolytic properties have been studied using both purified preparations of the 27 kDa .delta.-endotoxin and a 25 kDa segment thereof. See, Davidson et al., Curr. Microbiol., 11: 171-174 (1984); Thomas, W. E. , Ph.D., Thesis, University of Cambridge, "Biochemistry and Mode of Action of the Insecticidal .delta.-endotoxins of Bacillus thuringiensis" (1984); Armstrong, et al., J. Bacteriol., 161: 39-46 (1985); Wu et al., FEBS Letts., 190: 232-236 (1985); Lee et al., Biochem. Biophys. Res. Commun., 126: 953-960 (1985); Hurley et al., Biochem. Biophys. Res. Commun., 126: 961-965 (1985); Sriram et al., Biochem. Biophys. Res. Commun., 132: 19-27 (1985); and Cheung et al., Curr. Microbiol., 12: 121-126 (1985).
Using a somewhat different approach to investigate the properties of this polypeptide, the gene encoding the 27 kDa .delta.-endotoxin has been cloned in both Escherichia coli (see, Ward et al., FEBS Letts., 175: 377-781 (1984); Waalwijk et al., Nucleic Acids Res., 13: 8201-8217 (1985); Bourgouin et al., Mol. Gen. Genet., 205: 390-397 (1986), and in sporogenic and asporogenic strains of Bacillus subtilis (see, Ward et al., J. Mol. Biol., 191: 1-11 (1986); Ward et al., J. Mol. Biol., 191: 13-22 (1986)). In E. coli, induction of a high level of wild type 27 kDa .delta.-endotoxin expression has been found to have a significant deleterious effect on the growth of that bacterium. It has been postulated that the observed deleterious effect is due to binding of the toxin to phosphatidyl choline and phosphatidyl ethanolamine lipid receptors in E. coli cell plasma membranes. See, Ward, E. S., Ph.D. Thesis, University of Chambridge, "Molecular Genetics of an Insecticidal .delta.-endotoxin from Bacillus thuringiensis var. israelensis" (1988); Thomas, et al., FEBS Letters, 154: 362-368 (1983). In B. subtilis recombinants, the 27 kDa protein accumulates in the cytoplasm as phase bright crystalline inclusions, similar in appearance, but smaller than, the var. israelensis crystal. These inclusions have been purified and shown to consist entirely of 27 kDa .delta.-endotoxin. See, Ward et al., J. Mol. Biol., 191: 1-11 (1986); Ward et al., J. Mol Biol., 191: 13-22 (1986). Testing these inclusions in the absence of other crystal polypeptides, has shown this polypeptide to be both larvicidal and haemolytic. See, Ward et al., J. Mol. Biol., 191: 1-11 (1986); Ward et al., J. Mol. Biol., 191: 13-22 (1986). These results are consistent with those reported by Davidson et al., Curr. Microbiol., 11: 171-174 (1984), Thomas, W. E., Ph.D. Thesis, University of Cambridge, "Biochemistry and Mode of Action of the Insecticidal .delta.-endotoxins of Bacillus thuringiensis" (1984), and Armstrong et al., J. Bacteriol., 161: 39-46 (1985), but differ from those of several other groups who did not detect mosquitocidal activity in their preparations of this protein, such as Wu et al., FEBS Letts. 190: 232-236 (1985), Lee et al., Biochem. Biohphys. Res. Commun., 126: 953-960 (1985), Hurley et al., biochem. Biophys. Res. Commun. 126: 961-965 (1985), and Cheung et al., Curr. Microbiol., 12: 121-126 (1985).
The nucleotide sequence of the 27 kDa .delta.-endotoxin has been reported in the literature. See, Waalwijk et al., Nucleic Acids Res., 13: 8207-8217 (1985); Ward et al., J. Mol. Biol., 191: 1-11 (1986). the hytdropathyl plot of this protein shows it to be highly hydrophobic, and the protein has been shown to interact with specific plasma membrane phospholipids. See, Thomas et al., FEBS Letts., 145: 362-368 (1983). It has also recently been shown by Knowles et al. Biochem. Biophys. Acta., 924: 509-518 (1987) that this protein shares a common cytolytic mechanism with other B. thuringiensis .delta.-endotoxine from other serotypes. Commentators in this field have theorized that these .delta.-endotoxins bind to receptors on the membrane, and subsequently interact with the membrane to create a hole or pore. The generation of these pores is thought to lead to colloid-osmotic lysis, where an inflow of ions si accompanied by water influx, which in turn causes cell swelling followed by lysis. See, Knowles et al. Biochem. Biophys. Acta., 924: 509-518 (1987).
The present invention is based on a more detailed understanding of the interaction of the var. israelensis 27 kDa .delta.-endotoxin with target membranes. Through in vitro mutagenesis techniques, specific codon alterations have been directed in the cloned .delta.-endotoxin gene. Various of the mutant proteins have been found to possess greater solubility characteristics, less haemolytic activity, and/or greater expression potential in cells containing significant amounts of phosphatidate-type toxin receptors, than the protein encoded by the wild type 27 kDa Bacillus thuringiensis var. israelensis gene.